Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

Morphological Changes of Hemocytes of Musca domestica Larva in vitro Infected by Escherichia coli

[Objective] The research aimed to observe the effects of Escherichia coli infection on the morphology of hemocytes of the 3rd stage larva of Musca domestica in vitro and understand the hemocytes types that take part in the cell immunity of Musca domestica larval.[Method] The hemcytes of the 3rd stage larva of Musca domestica were cultured in vitro and the hemocyte morphology was observed about 2,4,6,8 h after culture in vitro.After Escherichia coli were injected into the hemocytes of the 3rd stage larva of Musca domestica in vitro,the morphology changes of hemocytes were observed at different time after infection.[Result] The hemocytes of of the 3rd stage larva of Musca domestica was divided into five types about 2 h after hemoculture.The hemocytes partly adherence was seen about 6 h after hemoculture.The vacuolation and morpholysis was found in plasmatocytes after being infected by E.coli and a great quantity bacterium were gathered around granulocyte,but the morphology changes of hemocytes were not found in the prohemocyte,shprulocyte and oenocytoid.[Conclusion] The plasmatocyte and granulocyte were primary participants of the cell immunity of Musca domestica larval,but the prohemocyte,sphrulocyte and oenocytoid do not participate in the cell immune reactions.

Effects of Destruxin A on Hemocytes Morphology of Bombyx mori

Destruxin A （DA）, a kind of cyclo-hexadepsipeptide isolated from entomopathogenic fungus, Metarhizium anisopliae, is an inhibitor of insect＇s immunity. But its mechanism has not been clarified yet. In this study, the effects of DA on morphologic changes of in vivo and in vitro hemocytes of silkworm, Bombyx mori, were investigated by means of inverted phase contrast microscopy （IPCM）, fluorescence microscopy （FCM） and environmental scanning electron microscopy （ESEM）. The results indicated that DA was cytotoxic to granulohemocytes （GR） and plasmatocytes （PL）. The LC 50 values of DA against in vitro GR and PL of silkworm were 68.77 and 84.11 μg mL-1, respectively. However, the hemocytes in vivo were more susceptible to DA, although at the extremely low dose of 10 μL of 12.5 μg mL-1 for each insect （i.e., 0.036 μg g-1 body weight, or approximately 0.25 μg mL-1 hemolymph）, DA could induce obviously morphologic alterations of hemocytes in vivo. The results imply that there might be some factors in silkworm＇s hemolymph, which influence the interaction of DA and hemocytes.

Effects of Several Immunostimulants on Phenoloxidase and Hemocytes of the Crab Charybdis japonica

To investigate the stimulating effects of immunostimulants on the autogenous immunocompetence of crabs and the possible mechanisms involved, the immunostimulating effects of β-1,3-glucan, lipopolysaccharide (LPS), inactivated Vibrio harveyi and Vibrio anguillarum on phenoloxidase (PO) and hemocytes of Charybdis japonica were investigated in this study. It was found that the yields and the enzymatic activities of purified PO in C. japonica increased significantly after the crabs were treated with immunostimulants, while the unit enzymatic activities remained almost the same. After treatment with β-1,3-glucan and LPS, the amount of rough endoplasmic reticulum (RER) and the number of mitochondria in both semigranular cells and granular cells increased greatly, and the number of cytoplasmic granules decreased but with enlarged volume. However, the corresponding characteristics of hyaline cells remained almost the same. On the other hand, the number of granules in semigranular cells decreased greatly, and the number of mitochondria of hyaline cells increased greatly, after treatment with inactivated vibrios. It may be concluded that the effect of polysaccharide immunostimulants on the innate immune system of C. japonica is different from that of inactivated vibrio immu-nostimulants. The immunity-enhancing mechanism of polysaccharides in crab autogenous immunocompetence is probably accomplished by the increased yields of PO and total PO activities, while that of inactivated vibrios is probably accomplished by the partially increased yields of PO and total PO activities as well as the significantly improved phagocytotic abilities of semigranular cells and hyaline cells.

Early Detection of White Spot Syndrome Virus(WSSV)in Isolated Hemocytes of Litopenaeus vannamei

To date, White Spot Syndrome (WSS) produced by the White Spot Syndrome Virus (WSSV) causes one of the most severe diseases infecting penaeid shrimps worldwide. Although a vast amount of studies has elucidated pathogenesis in live infection models, there is still little information about the interaction of WSSV infections using in vitro models in the whiteleg shrimp Litopenaeus vannamei (L. vannamei) hemocytes. In this study, a WSSV infection kinetics was performed using total hemocytes isolated from healthy L. vannamei organisms and maintained in in vitro conditions using isotonic solution for shrimp (ISS). The infected experimental cells received ≈ 30,000 viral copies of WSSV. The viability of the hemocytes (control and infected group) was measured during the kinetics with trypan blue exclusion method and cells were maintained up to 6 hpi (post-infection) with non-significant differences of viability between both groups. WSSV replication was assessed using RT- PCR at the RNA expression level of the early viral gene Ie1 and transcripts were detected as early as 30 min pi. Hemocytes from WSSV group showed disrupted integrity, degranulation and irregular shape. This study provides evidence of the capability of WSSV to infect and replicates in L. vannamei hemocytes using in vitro assays in short times as 30 min.

Effects of mosquito hemocytes on normal and degenerated oocysts of Plasmodium yoelii and their role in oocyst melanization

Anopheles stephenst, infected with Plasinodium yoelii, was fed with 10% sucrose solution contatning 1% difluoromethylornithine (DFMO) to induce oocyst degeneration of the malaria parasites. On the 11th day after infection, the effects of mosquito hemocytes On the normal and degenerated oocysts were observed under transmission electron microscope. In the control group, no hemocyte could be found around the normally-developed oocysts. In the DFMO-treated group, all the oocysts underwent degeneration in various degrees and some of them were melanized. All the oocysts were attached by one or more hemocytes of the kind of granulocytes.There were many granules with microtubular structure in the cytoplasm of the hemocytes and in the space between the hemocyte and the oocyst. The findings suggest that the degeneration of oocysts can attract the hemocytes to attach around them and the latter can release granules and possibly other substances to cause encapsulation and melanization of the oocvsts.

ON THE ULIRASTRUCTURE AND CLASSIFICATION OF THE HEMOCYTES OF PENAEID SHRIMP, PENAEUS VANNAMEI (CRUSTACEA, DECAPODA)

The ultrastructure of hemocytes of Penaeus vannamei was examined by transmissionelectron mircroscope. Three types of hemocytes were identified: hyaline cell, small-granule cell andlarge-granule cell. Hyaline cells are the smallest of the hemocytes, lack electron-dense granules, haveaverage size of 11. 11 μp (SE ± 0.50, n = 10) × 6.00 μp (SE ± 0.55, n = 10), comprise 25. 8%(SE ± 2.87, n = 4 ×10) of the hemocyt; small granule cells are the mos abundan type of hamocytes,cornprise 58. 1% (SE ± 3.4o, n = 4 ×10) of the hees, contaln sInall electron - dense ,haVe average sbe of 10.78 mp (SE ± 0.00, n = 10) × 8.63 mp (SE ± 0.44, n = 10); and largn-gran-ule cells comprise 16. 1% (SE ± 2. 55, n = 4 × 10) of the hemocytes, are filled with large elecbondense guules, have average size of 12.51 μp (SE ± 0.63, n = 10) × 8.99μm (SE ±0.71, n = 10).

Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects,the cell-mediated immune response involves an active role of hemo-cytes in phagocytosis,nodulation,and encapsulation.Although these processes have been well documented in multiple species belonging to different insect orders,information con-cerning the immune response,particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens,is still limited.This is a serious gap in knowledge given the high economic relevance of H.illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections.The present study repre-sents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect.In particular,in addition to prohemocytes,we identified five hemocyte types that mount the immune response in the larva,and analyzed their behavior,role,and morphofunctional changes in response to bac-terial infection and injection of chromatographic beads.Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes.These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells,devel-oping a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph,respectively.These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes,which could thus trophically support other hemocytes during the immune response.Finally,the regulation of the cell-mediated im-mune response by eicosanoids was investigated.

Parasitism of Pieris rapae （Lepidoptera： Pieridae） by the endoparasitic wasp Pteromalus puparum （Hymenoptera： Pteromalidae）： Effects of parasitism on differential hemocyte counts, micro- and ultra-structures of host hemocytes

Parasitism by the endoparasitic wasp Pteromalus puparum （Hymenoptera： Pteromalidae） by using only its associated venom, can suppress the immunal responses of Pieris rapae （Lepidoptera： Pieridae）. However, up to now, current knowledge of the mech- anisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy （TEM）. Five hemocyte types, prohemocytes （PRs）, granulocytes （GRs）, plasmatocytes （PLs）, oenocytoids （OEs） and coagulocytes （COs）, were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.

Hemocytes transcriptome profile of the Chinese mitten crab(Eriocheir sinensis)

The Chinese mitten crab(Eriocheir sinensis)possesses an open circulatory system,in which hemolymph moves through interconnected sinuses or spaces surrounding organs called hemocoels.The hemocytes are classified into hyalinocytes,semigranulocytes,and granulocytes;and limited transcriptomics research is available.In this study,the transcriptome of the crab E.sinensis hemocytes was characterized.A total of 14,380,229 clean reads representing a total of 4.31 Gb nucleotides dataset were produced.A total of 67,047 contigs were obtained and 12.49%(8375)and 9.74%(6533)of the contigs were matched to data publicly available from the GenBank nr nucleotide and Uniprot databases,respectively.Among these contigs,4344 contigs belong to three categories of Gene Ontology,126 contigs to 21 subcategories of KEGG,and 4962 contigs to 25 categories of the COG database.A total of 508,379 and 345 transcripts of the E.sinensis showed>40%sequence similarity with transcripts expressed in the vertebrate blood cells from tilapia(Oreochromis niloticus),Chinese softshell turtle(Trionyx sinensis)and chicken(Gallus gallus)respectively.A total of 53,077 single nucleotide polymorphisms were identified and 9912 SSRs for microsatellite mining were found.The most frequent repeat motifs detected were dinucleotide repeats,accounting for 37.52%of the total repeats.Our data provides an important gene resource for the exploitation of molecular marker-assisted breeding,immune mechanisms and conservation of germplasm in the crab E.sinensis.

Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx moil

Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace＇s medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs ofpndpS. Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others.

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry

Single-cell mass cytometry(SCMC)combines features of traditional flow cytometry(i.e.,fluorescence-activated cell sorting)with mass spectrometry,making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms.In this study,we optimized SCMC to analyze hemocytes of the Drosophila innate immune system.We used metal-conjugated antibodies(against cell surface antigens H2,H3,H18,L1,L4,and P1,and intracellular antigens 3A5 and L2)and anti-IgM(against cell surface antigen L6)to detect the levels of antigens,while anti-GFP was used to detect crystal cells in the immune-induced samples.We investigated the antigen expression profile of single cells and hemocyte populations in naive states,in immune-induced states,in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase(hopTum)and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1[l(3)mbn1],as well as in stem cell maintenance-defective hdcD84 mutant larvae.Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes,plasmatocytes,and crystal cells,and delineated the unique immunophenotype of Drosophila mutants.We have identified subpopulations of L2^(+)/P1^(+)and L2^(+)/L4^(+)/P1^(+)transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum,respectively,and a subpopulation of L4^(+)/P1^(+)cells upon immune induction.Our results demonstrated for the first time that SCMC,combined with multidimensional bioinformatic analysis,represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

Development of two continuous hemocyte cell sublines in the Asian corn borer Ostrinia furnacalis and the identification of molecular markers for hemocytes

Granulocytes and plasmatocytes play important roles in clearing foreign objects in insects,but it is difficult to distinguish between them in immune reactions.Based on the hemocyte cell line SYSU-OfHe-C established at our lab,two cell sublines,SYSU-OfHe-C Granulocyte(Gr cells)and SYSU-OHe-C Plasmatocyte(PI cells),which possess the morphological characteristics of granulocytes and plasmatocytes,respectively,were established.Gr and PI cells showed different behaviors in immune reactions,such as spreading,phagocytosis and encapsulation.PI cells were easier to spread,but Gr cells tended to undergo aggregation,indicating that they may take different strategies to clear foreign objects.These results also suggested that granulocytes and plasmatocytes may express some different proteins.By comparing the gene expression in cells from the two sublines,1662 differentially expressed genes were identified,and 13 out of 30 transmembrane proteins highly ex pressed in PI cells(six)or Gr cells(seven)were further screened and confirmed by reverse-transcription polymerase chain reaction(PCR).Finally,three transmembrane genes specifically expressed in Pl cells and two transmembrane genes specifically expressed in Gr cells were screened out based on their expressions in immune reactions by quantitative PCR analysis.These genes may potentially be used as molecular markers to distinguish between granulocy tes and plasmatocytes in Ostrinia fiurnacalis,and further to clarify the functions of immune hemocytes in cellular immune reaction such as encapsulation and so on.

Characterization and mode of action analysis of black soldier fly(Hermetia illucens)larva-derived hemocytes

With the growing importance of the black soldier fly(Hermetia illucens)for both sustainable food production and waste management as well as for science,a great demand of understanding its immune system arises.Here,we present the first description of the circulating larval hemocytes with special emphasis on uptake of microorganisms and distinguishing hemocyte types.With histological,zymographic,and cytometric methods and with a set of hemocyte binding lectins and antibodies,the hemocytes of H.illucens are identified as plasmatocytes,crystal cells,and putative prohemocytes.Total hemocyte counts(THC)are determined,and methods for THC determination are compared.Approximately 1100 hemocytes per microliter hemolymph are present in naive animals,while hemocyte density decreases dramatically shortly after wounding,indicating a role of hemocytes in response to wounding(and immune response in general).The determination of the relative abundance of each hemocyte type(differential hemocyte count,DHC)revealed that plasmatocytes are highly abundant,whereas prohemocytes and crystal cells make up only a small percentage of the circulating cells.Plasmatocytes are not only the most abundant but also the professional phagocytes in H.illucens.They rapidly engulf and take up bacteria both in vivo and in vitro,indicating a very potent cellular defense against invading pathogens.Larger bioparticles such as yeasts are also removed from circulation by phagocytosis,but slower than bacteria.This is the first analysis of the potent cellular immune response in the black soldier fly,and a first toolbox that helps to identify hemocyte(types)is presented.

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.

Effect of E. coli coli on Anti-disease Activities of Scailops: Argopecten irradians

The effect of acute E. coli challenge on the anti-disease activity of scallops Argopecten irra-dians is examined. The treatments of scallop from which hemolymph samples were taken for study included (1) control scallops, (2) sham-injected scallops, (3) PSW-injected scallops and (4) E. coli-injected scallops. From the beginning, the anti-disease activities of scallops are deter -mined at 12 hr and 24 hr.The concentrations of circulating hemocytes, the total serum protein concentrations and the activities of alkaline phosphatase, acid phosphatase and superoxide dismutase in the scallops Argopecten irradians are determined.Injection with E. coli results in a significant elevation in the concentration of circulating hemocytes and in the alkaline phosphatase activity and a significant decline in the total serum protein concentration and in the superoxide dismutase activity at 24 hr postchallenge. It shows that metabolism of bay scallop is expedited to adopt the challenge.

Effect of Dopamine Injection on the Hemocyte Count and Prophenoloxidase System of the White Shrimp Litopenaeus vannamei

Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase in-hibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured (P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups,the hemocyte count reached the minimum in 3 h;granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macro-globulin-like activity reached the maximum in 3 h,and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or sponta-neously activated under a normal physical condition.

Modulation by Biogenic Amines for the Hemocyte Count and Prophenoloxidase Exocytosis via Receptors in Litopenaeus vannamei

Hemocyte counts and phenoloxidase(PO)activity were examined after hemolymph being incubated in dopamine(DA),noradrenaline(NE)and serotonin(5-HT).Results showed that all the three biogenic amines(BAs)had a significant impact on total hemocyte count(THC),differential hemocyte count(DHC),and intracelluar and extracelluar phenoloxidase(PO)activity.Among these Bas,DA had the strongest effect on the above parameters,whereas 5-HT had the least effect.Preincubation with D1 receptor antagonist SCH23390,D2 receptor antagonist Sulpiride and 1:1 admixture of the two could significantly inhibit the effect of DA on these parameters.SCH23390 showed a stronger inhibitory effect than Sulpiride,and the admixture exhibited the strongest effect.These results suggested that the change of hemocyte count and activation of prophenoloxidase(proPO)system in Litopenaeusvan-namei hemocyte can be regulated by BAs,and DA modulates the two parameters via its receptors.

Hematological Changes in White Spot Syndrome Virus-Infected Shrimp,Fenneropenaeus chinensis(Osbeck)

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV(white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifesta-tions of hemocytes were similar among moribund shrimps infected via injection,feeding and by nature. Firstly,the total hemocyte counts(THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly,necrotic,broken and dis-integrated cells were often observed,and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly,necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared,whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV,and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.